Note: Descriptions are shown in the official language in which they were submitted.
CA 02701571 2010-04-01
Polypeptide Markers for the Diagnosis of Prostate Cancer
The present invention relates to the measuring of one or more peptide markers
in
a sample from a subject for the diagnosis of prostate diseases (PCA) and to a
method for the diagnosis of prostate cancer, wherein the presence or absence
of
the peptide marker or markers is indicative of the existence of prostate
cancer.
The carcinoma of the prostate gland is one of the most common cancers in
males.
Since complaints occur only in a stage of advanced disease, the cancer can be
diagnosed in an early stage only by regular screening tests for early
detection
(digital rectum exam and PSA (prostate specific antigen) value in the blood).
To
confirm the suspicion diagnosis, a tissue specimen is withdrawn by means of
fine-
needle biopsy.
For the therapy, there are several possibilities that depend on the kind and
stage
of the tumor and on the individual needs of the patient: In an early stage,
seed
implantation (minimal-invasive introduction of iodine-125 radioactive emitters
into
the prostate) or surgical removal of the tumor and irradiation from outside
are
available.
At the time of diagnosis, metastasizing into other organs has already occurred
in
one third of the patients; at this time, the disease can hardly be healed any
more.
However, radiotherapy, chemotherapy or hormone therapy (combinations are
possible) can delay the further spreading of the cancer. When the prostate
gland is
affected isolatedly, i.e., metastasizing has not occurred yet, the prognosis
is
favorable.
A benign tumor (adenoma) of the prostate gland, benign prostate hyperplasia
(BPH), occurs much more frequently than the carcinoma. According to the prior
art, it can be distinguished from a malignant carcinoma only very unreliably
by
means of the PSA value. In this case too, a biopsy must be effected to be able
to
make a clear diagnosis.
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As described above, there is no non-invasive and reliable early detection of
prostate cancer. To date, a clear diagnosis has been associated with invasive
operations, such as biopsy. Thus, there has been a need to find a process and
method for a diagnosis of prostate cancer that is as little as possible
invasive,
quick and inexpensive.
WO 03/072710 describes protein biomarkers for distinguishing between prostate
carcinoma cells and BPH. The application describes markers that have high
uncertainties (in a range of more than 0.5%). Due to the imprecise
information,
an assignment to individual markers is virtually impossible from the large
number
of molecules in this range. A cell lysate of prostate epithelial cells is
preferably
employed as a sample material, which requires a complicated sampling
procedure.
Due to the large number of proteins included in the definition and the
difficulty of
sampling, the method is little suitable.
WO 01/25791 describes methods for the diagnosis of prostate carcinomas using
markers. The mentioned markers are derived from the protein semenogelin I. The
stated masses are defined to a certainty of only 0.5%. Studies show that the
mentioned markers are unsuitable.
WO 2006/106129 describes polypeptide markers for the diagnosis of prostate
cancer from urine samples, among others.
When this method was performed, it was found that the measurements of samples
from different subjects had large variabilities, and problems arose in the
evalua-
tion.
Surprisingly, it has now been found that there are markers that offer a better
significance under such circumstances, especially if they are employed in
combina-
tion with an altered sample preparation.
Surprisingly, it has now been found that particular peptide markers in a
sample
from a subject can be used for the diagnosis of prostate cancer and for the
differential diagnosis to distinguish between prostate cancer and benign
prostate
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hyperplasia (BPH). In particular, the samples may be urine or seminal fluid
samples, which are withdrawn non-invasively.
Consequently, the present invention relates to the use of the presence or
absence
of at least three polypeptide markers in a sample from a subject for the
diagnosis
of prostate diseases, wherein said polypeptide markers are selected from
polypep-
tide marker Nos. 1 to 141 as characterized by the molecular masses and
migration
times as stated in Table 1.
Table 1: Polypeptide markers for the diagnosis of prostate diseases and their
molecular masses and migration times (CE time in minutes):
Number Mass (Da) CE time [min]
1 858.4 23.3
2 911.5 25.9
3 1016.3 35.7
4 1016.5 25.8
1050.5 26.9
6 1068.6 21.7
7 1096.5 26.1
8 1128.5 25.7
9 1134.6 23.6
1154.6 25.7
11 1157.6 37.4
12 1179.6 27.1
13 1186.6 22.4
14 1191.6 36.1
1194.6 26.7
16 1200.6 24.2
17 1216.6 24.3
18 1225.6 26.3
19 1257.5 34.1
1265.6 27.1
21 1312.7 22.4
22 1358.4 36.5
23 1392.7 21.7
24 1449.7 21.8
1487.7 29.6
26 1523.7 22.0
27 1525.5 37.2
28 1552.6 37.2
29 1576.7 26.5
1576.7 46.0
31 1579.8 20.1
32 1584.6 37.7
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33 1588.8 30.2
34 1592.8 22.2
35 1600.6 37.9
36 1627.8 29.5
37 1631.8 47.0
38 1634.9 29.7
39 1636.8 22.5
40 1640.8 28.1
41 1649.8 22.6
42 1680.8 30.0
43 1684.7 31.5
44 1687.6 37.8
45 1706.9 22.7
46 1714.6 37.9
47 1725.7 38.4
48 1728.8 36.8
19 1731.8 22.7
50 1755 31.4
51 1769.8 28.2
52 1783.9 39.9
53 1794 32.4
54 1806.9 23.1
55 1813.8 31.7
56 1819.9 23.4
57 1825.9 20.1
58 1844.6 34.2
59 1854.9 41.4
60 1860.4 33.6
61 1878.7 30.9
62 1882.9 20.3
63 1911.1 25.1
64 1925.9 23.3
65 1945.1 33.7
66 1950.9 35.8
67 1955.9 28.1
68 1969.9 25.3
69 1993 27.1
70 2031 32.6
71 2133 25.9
72 2157.1 22.2
73 2168.9 33.9
74 2184.8 34.2
75 2188 39.9
76 2210.9 37.7
77 2282.1 34.0
78 2355.2 22.7
19 2356.8 35.5
80 2414.7 35.6
81 2483.2 27.7
82 2570.3 42.8
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83 2577.3 24.7
84 2599.3 28.0
85 2668.4 42.1
86 2682.2 22.5
87 2702.1 38.2
88 2726.4 43.2
89 2742.4 42.3
90 2751.5 29.2
91 2753.4 36.3
92 2754.3 29.7
93 2799.2 25.1
94 2854.5 34.9
95 2977.6 29.1
96 3001.5 35.5
97 3013.2 22.3
98 3021.5 23.5
99 3048 28.7
100 3092 29.7
101 3139.5 29.6
102 3145.6 38.8
103 3166.4 22.1
104 3168.4 24.7
105 3193.3 22.6
106 3256.6 33.1
107 3260.5 41.6
108 3292.7 39.5
109 3409.7 32.2
110 3425.7 31.3
111 3426.5 27.8
112 3530.6 26.2
113 3559.8 24.9
114 3657.8 40.7
115 3718.8 32.5
116 3734.9 32.5
117 3765.5 20.2
118 3775.7 25.9
119 3788.9 25.3
120 3858.9 25.8
121 3968.7 21.1
122 3986.8 20.6
123 3996.7 20.9
124 4043.7 20.4
125 4045 26.4
126 4098 24.6
127 4218 26.1
128 4353 20.2
129 4405 20.7
130 4410 20.0
131 4436.1 26.3
132 4672 23.3
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133 4863.3 26.7
134 5000.2 24.4
135 6169.9 24.6
136 8837.7 21.1
137 8854 21.1
138 9866.8 20.9
139 10342.3 23.0
140 10753.7 19.7
141 10770.2 19.6
With the present invention, it is possible to diagnose prostate cancer at a
very
early stage. Thus, the disease can be cured by known methods at an early
stage.
The invention further enables an inexpensive, quick and reliable diagnosis
with in
part non-invasive or only minimal-invasive operations.
The invention further relates to differential diagnosis for distinguishing
between
prostate carcinoma and BPH. The differential diagnosis can be effected by
using
the presence or absence of at least three polypeptide markers in a sample from
a
subject, wherein said polypeptide markers are selected from polypeptide
markers
142 to 201, which are characterized by the molecular masses and migration
times
as stated in Table 3. Preferably, more markers are employed.
Table 3: Polypeptide markers for the differential diagnosis of prostate cancer
or
BPH, their molecular masses and migration times.
No.
Mass (Da) CE time (min)
142 1210.4 36.5
143 1210.6 20.9
144 1234.6 27.4
145 1235.6 26.7
146 1268.6 21.4
147 1276.5 36.0
148 1390.5 37.1
149 1440.6 24.3
150 1467.7 21.7
151 1491.8 39.9
152 1495.6 37.4
153 1510.7 24.3
154 1523.8 40.5
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155 1552.6 37.2
156 1579.8 29.8
157 1584.6 37.7
158 1624.6 37.7
159 1664.8 29.8
160 1680.8 30.0
161 1687.6 37.8
162 1706.9 22.7
163 1714.6 37.9
164 1725.7 38.4
165 1783.9 39.9
166 1794.9 24.0
167 1822.8 30.9
168 1825.9 20.1
169 1864.0 44.1
170 1882.9 20.3
171 1925.9 23.3
172 1964.0 31.8
173 2089.0 23.7
174 2118.0 33.0
175 2133.0 25.9
176 2170.0 33.4
177 2189.1 26.8
178 2282.1 34.0
179 2298.1 33.9
180 2442.2 34.1
181 2485.2 34.4
182 2599.3 28.0
183 2686.9 29.1
184 2695.3 23.5
185 2839.0 24.2
186 3092.0 29.7
187 3121.4 30.3
188 3248.5 30.7
189 3302.8 23.2
190 3333.8 23.8
191 3409.7 32.2
192 3425.7 31.3
193 3457.7 31.5
194 3478.4 41.8
195 3524.6 32.4
196 3589.8 25.1
197 3765.5 20.2
198 3839.9 19.7
199 4290.0 28.8
200 6650.9 25.6
201 10770.2 19.6
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The migration time is determined by capillary electrophoresis (CE), for
example, as
set forth in the Example under item 2. In this Example, a glass capillary of
90 cm
in length and with an inner diameter (ID) of 50 pm and an outer diameter (OD)
of
360 pm is operated at an applied voltage of 30 W. As the mobile solvent, 30%
methanol, 0.5% formic acid in water is used.
It is known that the CE migration times may vary. Nevertheless, the order in
which
the polypeptide markers are eluted is typically the same under the stated
condi-
tions for any CE system employed. In order to balance any differences in the
migration time that may nevertheless occur, the system can be normalized using
standards for which the migration times are exactly known. These standards may
be, for example, the polypeptides stated in the Examples (see the Example,
item
3).
The characterization of the polypeptides shown in Tables 1 and 3 was
determined
by means of capillary electrophoresis-mass spectrometry (CE-MS), a method
which
has been described in detail, for example, by Neuhoff et al. (Rapid
communications
in mass spectrometry, 2004, Vol. 20, pages 149-156). The variation of the
molecular masses between individual measurements or between different mass
spectrometers is relatively small when the calibration is exact, typically
within a
range of 0.03%, preferably within a range of 0.01%.
The polypeptide markers according to the invention are proteins or peptides or
degradation products of proteins or peptides. They may be chemically modified,
for
example, by posttranslational modifications, such as glycosylation,
phosphoryla-
tion, alkylation or disulfide bridges, or by other reactions, for example,
within the
scope of degradation. In addition, the polypeptide markers may also be
chemically
altered, for example, oxidized, during the purification of the samples.
Proceeding from the parameters that determine the polypeptide markers (molecu-
lar weight and migration time), it is possible to identify the sequence of the
corresponding polypeptides by methods known in the prior art.
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The polypeptides according to the invention are used to diagnose prostate
diseas-
es, especially prostate cancer. "Diagnosis" means the process of knowledge
gaining by assigning symptoms or phenomena to a disease or injury. In the
present case, the existence of prostate cancer is concluded from the presence
or
absence of particular polypeptide markers. Thus, the polypeptide markers
accord-
ing to the invention are determined in a sample from a subject, wherein its
presence or absence allows to conclude the existence of prostate cancer in the
case of frequency markers. The presence or absence of a polypeptide marker can
be measured by any method known in the prior art. Methods which may be used
are exemplified below.
A polypeptide marker is considered present if its measured value is at least
as high
as its threshold value. If the measured value is lower, then the polypeptide
marker
is considered absent. The threshold value can be determined either by the
sensitivity of the measuring method (detection limit) or defined from
experience.
In the context of the present invention, the threshold value is considered to
be
exceeded preferably if the measured value of the sample for a certain
molecular
mass is at least twice as high as that of a blank sample (for example, only
buffer
or solvent).
The polypeptide marker or markers is/are used in such a way that its/their
presence or absence is measured, wherein the presence or absence is indicative
of
prostate diseases, especially prostate cancer (frequency markers). Thus, there
are
polypeptide markers which are typically present in patients with prostate
diseases,
but absent or less frequent in subjects with no prostate cancer (control). In
addition, there are polypeptide markers which are present in subjects with no
prostate cancer, but are less frequently or not at all present in subjects
with
prostate cancer.
In addition or also alternatively to the determination of the presence or
absence,
the amplitudes may also be used for the diagnosis of prostate diseases.
Amplitude
markers are used in such a way that the presence or absence is not critical,
but
the height of the signal (the amplitude) decides if the signal is present in
both
CA 02701571 2010-04-01
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groups. In Tables 2 and 4, the mean amplitudes of the corresponding signals
(characterized by mass and migration time) averaged over all samples measured
are stated. To achieve comparability between differently concentrated samples
or
different measuring methods, all peptide signals of a sample are normalized to
a
total amplitude of 1 million counts. Therefore, the respective mean amplitudes
of
the individual markers are stated as parts per million (ppm). All groups
employed
consist of at least 20 individual patient or control samples in order to
obtain a
reliable mean amplitude. The decision for a diagnosis is made as a function of
how
high the amplitude of the respective polypeptide markers in the patient sample
is
in comparison with the mean amplitudes in the control group or the "prostate
group". If the amplitude rather corresponds to the mean amplitudes of the
prostate group, the existence of a prostate disease is to be considered, and
if it
rather corresponds to the mean amplitudes of the control group, the non-
existence
of a prostate disease is to be considered.
The smaller the distance between the amplitudes of the control group and the
prostate group, the closer a value that lies between the two reference values
has
to be to one of the reference values.
One possibility is to subdivide the range between the mean amplitudes into
three
portions. If the value is in the lower third, this is indicative of the lower
value; if
the value is in the upper third, this is indicative of the upper value. If it
is in the
middle third, a definite statement about this marker is not possible.
Table 2: Amplitude markers
Pathological prostate gland Physiological prostate gland
No. Mass (Da) CE time (min) Frequency Mean amp. Frequency Mean amp.
1 858.4 23.3 0.56 45 0.77 123
2 911.5 25.9 0.73 106 0.86 260
3 1016.3 35.7 0.96 497 0.90 947
1016.5 25.8 0.95 385 0.97 941
1050.5 26.9 0.75 103 0.90 356
1068.6 21.7 0.54 62 0.78 225
7 1096.5 26.1 0.89 1657 0.96 3708
8 1128.5 25.7 0.58 60 0.80 158
9 1134.6 23.6 0.60 67 0.80 279
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1154.6 25.7 0.57 95 0.80 252
11 1157.6 37.4 0.88 457 0.89 1152
12 1179.6 27.1 0.78 186 0.80 405
13 1186.6 22.4 0.76 169 0.82 386
14 1191.6 36.1 0.55 79 0.76 274
1194.6 26.7 0.94 924 0.98 1575
16 1200.6 24.2 0.99 2465 0.99 5193
17 1216.6 24.3 0.88 432 0.85 828
18 1225.6 26.3 0.87 155 0.85 256
19 1257.5 34.1 1.00 3161 0.96 2149
1265.6 27.1 0.86 2016 0.92 3539
21 1312.7 22.4 0.51 174 0.81 729
22 1358.4 36.5 0.99 1848 0.95 1405
23 1392.7 21.7 1.00 3689 0.97 2739
24 1449.7 21.8 1.00 4310 0.97 3020
1487.7 29.6 0.88 418 0.51 234
6 1523.7 22.0 0.95 4303 0.96 3040
7 1525.5 37.2 1.00 2319 0.93 1817
28 1552.6 37.2 0.99 2766 0.91 1862
29 1576.7 26.5 0.99 1736 0.93 921
1576.7 46.0 0.92 2959 0.57 1199
31 1579.8 20.1 0.98 6547 0.98 4228
32 1584.6 37.7 0.87 382 0.76 300
33 1588.8 30.2 0.94 596 0.46 114
34 1592.8 22.2 0.83 480 0.52 201
1600.6 37.9 0.88 388 0.76 232
6 1627.8 29.5 0.81 253 0.67 158
37 1631.8 47.0 0.96 11650 0.65 4248
38 1634.9 29.7 0.85 522 0.67 272
39 1636.8 22.5 1.00 10631 0.98 7771
0 1640.8 28.1 0.84 162 0.41 50
1 1649.8 22.6 0.93 699 0.82 422
2 1680.8 30.0 0.89 2045 0.86 1483
3 1684.7 31.5 0.99 2814 0.98 1441
4 1687.6 37.8 0.93 217 0.80 154
5 1706.9 22.7 1.00 796 0.93 408
6 1714.6 37.9 0.71 116 0.45 48
7 1725.7 38.4 1.00 2078 0.90 1407
8 1728.8 36.8 0.79 221 0.67 124
19 1731.8 22.7 0.88 172 0.69 108
50 1755 31.4 1.00 9478 0.92 3966
51 1769.8 28.2 0.93 403 0.69 137
52 1783.9 39.9 0.76 240 0.40 112
53 1794 32.4 0.88 287 0.78 202
54 1806.9 23.1 0.83 188 0.60 112
55 1813.8 31.7 0.99 2221 0.95 983
56 1819.9 23.4 1.00 3799 0.93 2664
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57 1825.9 20.1 0.95 1494 0.90 926
58 1844.6 34.2 0.88 326 0.54 137
59 1854.9 41.4 0.95 7712 0.84 4491
0 1860.4 33.6 0.85 401 0.64 194
1 1878.7 30.9 0.84 963 0.69 534
2 1882.9 20.3 1.00 18028 0.98 12004
3 1911.1 25.1 0.99 62532 0.96 39024
4 1925.9 23.3 0.75 120 0.28 44
1945.1 33.7 0.86 197 0.74 180
6 1950.9 35.8 0.97 593 0.77 357
7 1955.9 28.1 0.82 345 0.44 86
8 1969.9 25.3 0.89 915 0.61 242
9 1993 27.1 0.80 104 0.34 33
0 2031 32.6 0.92 838 0.80 538
11 2133 25.9 0.98 714 0.70 278
2 2157.1 22.2 0.79 347 0.90 736
13 2168.9 33.9 0.99 706 0.91 493
14 2184.8 34.2 0.95 610 0.88 300
2188 39.9 0.97 1452 0.81 606
6 2210.9 37.7 0.84 274 0.64 183
7 2282.1 34.0 0.99 5409 0.99 5449
8 2355.2 22.7 0.99 554 0.97 956
79 2356.8 35.5 0.98 571 0.75 230
80 2414.7 35.6 0.78 471 0.61 164
81 2483.2 27.7 0.76 155 0.84 353
82 2570.3 42.8 0.99 5993 0.80 3366
83 2577.3 24.7 0.96 297 0.82 516
84 2599.3 28.0 0.95 1856 0.79 180
85 2668.4 42.1 0.90 529 0.72 333
86 2682.2 22.5 0.99 844 0.91 588
87 2702.1 38.2 0.82 332 0.64 137
88 2726.4 43.2 0.99 4394 0.80 2280
89 2742.4 42.3 0.87 592 0.50 188
90 2751.5 29.2 0.87 239 0.55 139
91 2753.4 36.3 0.86 150 0.64 79
92 2754.3 29.7 0.88 318 0.77 182
93 2799.2 25.1 1.00 3236 1.00 2169
94 2854.5 34.9 0.99 3786 0.97 2215
95 2977.6 29.1 0.84 179 0.47 66
96 3001.5 35.5 0.99 12587 0.98 7515
97 3013.2 22.3 0.91 1824 0.98 3609
98 3021.5 23.5 1.00 1642 0.85 1055
99 3048 28.7 0.82 230 0.72 112
100 3092 29.7 0.99 780 0.89 618
101 3139.5 29.6 0.97 1549 0.86 673
102 3145.6 38.8 0.97 1147 0.84 663
103 3166.4 22.1 0.82 409 0.51 159
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104 3168.4 24.7 0.82 211 0.91 477
105 3193.3 22.6 0.81 383 0.88 783
106 3256.6 33.1 0.90 1008 0.71 564
107 3260.5 41.6 0.86 371 0.55 205
108 3292.7 39.5 0.99 5967 0.91 3920
109 3409.7 32.2 0.89 386 0.84 287
110 3425.7 31.3 1.00 1098 0.97 1200
111 3426.5 27.8 0.81 136 0.45 45
112 3530.6 26.2 0.95 775 0.73 317
113 3559.8 24.9 0.96 896 0.80 633
114 3657.8 40.7 0.96 964 0.80 648
115 3718.8 32.5 0.97 364 0.95 695
116 3734.9 32.5 0.91 200 0.93 410
117 3765.5 20.2 0.60 199 0.26 40
118 3775.7 25.9 0.99 1537 0.88 1110
119 3788.9 25.3 0.78 258 0.57 144
120 3858.9 25.8 0.97 975 0.82 690
121 3968.7 21.1 0.95 1430 0.83 904
122 3986.8 20.6 0.99 4017 0.86 1722
123 3996.7 20.9 0.83 707 0.39 197
124 4043.7 20.4 0.92 1038 0.67 496
125 4045 26.4 0.98 2449 0.88 1561
126 4098 24.6 0.94 370 0.90 780
127 4218 26.1 0.97 4520 0.88 2787
128 4353 20.2 0.98 4815 0.88 2186
129 4405 20.7 0.86 704 0.59 244
130 4410 20.0 0.82 997 0.60 472
131 4436.1 26.3 0.95 2663 0.87 1538
132 4672 23.3 0.90 309 0.77 196
133 4863.3 26.7 0.61 158 0.76 389
134 5000.2 24.4 0.99 2538 0.98 1743
135 6169.9 24.6 0.78 444 0.46 282
136 8837.7 21.1 0.94 1669 0.71 1430
137 8854 21.1 0.63 441 0.86 1383
138 9866.8 20.9 0.99 1874 0.83 944
139 10342.3 23.0 0.77 1771 0.35 279
140 10753.7 19.7 0.76 4585 0.33 247
141 10770.2 19.6 0.52 2190 0.14 59
For the differential diagnosis between PCA and BPH, Table 4 shows polypeptide
markers that are typically present in patients with prostate carcinoma, such
as the
marker No. 145, but are not or rarely present in subjects with BPH. Further,
there
are polypeptide markers that are present in subjects with BPH, but occur less
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frequently or not at all in subjects with PCA, for example, the polypeptide
marker
No. 163.
Table 4:
Mass (Da) CE time (min) PCa BPH
No. Frequency (%) Mean amp. Frequency (%) Mean amp.
142 1210.4 36.5 0.50 149 0.61 243
143 1210.6 20.9 0.65 121 0.54 101
144 1234.6 27.4 0.96 841 0.98 977
145 1235.6 26.7 0.69 550 0.55 458
146 1268.6 21.4 0.37 52 0.46 86
147 1276.5 36.0 0.98 2879 0.99 3241
148 1390.5 37.1 1.00 19627 0.97 21822
149 1440.6 24.3 0.66 135 0.62 107
150 1467.7 21.7 0.41 89 0.54 102
151 1491.8 39.9 0.68 217 0.76 269
152 1495.6 37.4 0.68 134 0.79 162
153 1510.7 24.3 0.39 47 0.50 77
154 1523.8 40.5 0.98 7023 0.98 7856
155 1552.6 37.2 0.98 2130 0.99 2530
156 1579.8 29.8 0.98 1824 0.99 2061
157 1584.6 37.7 0.82 301 0.89 397
158 1624.6 37.7 0.99 1020 0.99 1159
159 1664.8 29.8 0.92 386 0.94 475
160 1680.8 30.0 0.82 1626 0.92 2182
161 1687.6 37.8 0.88 161 0.88 193
162 1706.9 22.7 0.99 705 0.97 624
163 1714.6 37.9 0.52 72 0.67 105
164 1725.7 38.4 1.00 1646 0.99 1964
165 1783.9 39.9 0.63 175 0.73 224
166 1794.9 24.0 0.98 1179 0.96 1305
167 1822.8 30.9 0.67 192 0.59 148
168 1825.9 20.1 0.95 1629 0.96 1370
169 1864.0 44.1 0.70 874 0.77 1182
170 1882.9 20.3 0.99 18958 0.99 17650
171 1925.9 23.3 0.74 121 0.62 105
172 1964.0 31.8 0.54 96 0.42 71
173 2089.0 23.7 0.56 76 0.46 52
174 2118.0 33.0 0.43 42 0.55 61
175 2133.0 25.9 0.98 711 0.94 589
176 2170.0 33.4 0.34 201 0.48 295
177 2189.1 26.8 0.94 889 0.92 640
178 2282.1 34.0 1.00 5015 0.99 5720
179 2298.1 33.9 0.49 95 0.58 127
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180 2442.2 34.1 0.97 553 0.98 708
181 2485.2 34.4 0.49 42 0.57 67
182 2599.3 28.0 0.94 1303 0.93 1675
183 2686.9 29.1 0.54 102 0.66 117
184 2695.3 23.5 0.99 2295 1.00 2058
185 2839.0 24.2 0.77 589 0.86 687
186 3092.0 29.7 0.95 704 0.99 854
187 3121.4 30.3 0.93 585 0.94 690
188 3248.5 30.7 1.00 1184 1.00 1363
189 3302.8 23.2 0.65 102 0.68 135
190 3333.8 23.8 0.41 128 0.32 77
191 3409.7 32.2 0.81 357 0.92 508
192 3425.7 31.3 1.00 1132 1.00 1339
193 3457.7 31.5 1.00 13651 1.00 15121
194 3478.4 41.8 0.53 315 0.64 368
195 3524.6 32.4 0.49 285 0.61 397
196 3589.8 25.1 0.93 778 0.91 699
197 3765.5 20.2 0.64 200 0.43 132
198 3839.9 19.7 0.92 1868 0.92 1621
199 4290.0 28.8 0.97 2383 0.99 2698
200 6650.9 25.6 0.69 161 0.54 134
201 10770.2 19.6 0.44 1814 0.60 2746
The subject from which the sample in which the presence or absence or the
amplitude of one or more polypeptide markers is determined is derived may be
any subject which is capable of suffering from prostate diseases, for example,
an
animal or human. Preferably, the subject is a mammal, such as a dog or a
horse,
and most preferably, it is a human.
Preferably, for the application of the invention, not just one polypeptide
marker,
but a combination of markers are used to diagnose prostate cancer, wherein the
existence of prostate diseases is concluded from their presence or absence
and/or
the height of the amplitude. By comparing a plurality of polypeptide markers,
a
bias in the overall result from a few individual deviations from the typical
presence
probability in the sick or control individual can be reduced or avoided.
The sample in which the presence or absence or amplitude of the polypeptide
marker or markers according to the invention is measured may be any sample
which is obtained from the body of the subject. The sample is a sample which
has
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a polypeptide composition suitable for providing information about the state
of the
subject (prostate cancer or not). For example, it may be urine, sperm, seminal
fluid (sperm without spermatozoa). Preferably, it is a liquid sample.
In a preferred embodiment, the sample is a urine sample.
Urine samples can be taken as known in the prior art. Preferably, a first
stream or
midstream urine sample is used in the context of the present invention. For
example, the urine sample may be taken by means of a catheter or also by means
of an urination apparatus as described in WO 01/74275.
The presence or absence of a polypeptide marker in the sample may be deter-
mined by any method known in the prior art that is suitable for measuring
polypeptide markers. Such methods are known to the skilled person. In
principle,
the presence or absence of a polypeptide marker can be determined by direct
methods, such as mass spectrometry, or indirect methods, for example, by means
of ligands.
If required or desirable, the sample from the subject, for example, the urine
sample, may be pretreated by any suitable means and, for example, purified or
separated before the presence or absence of the polypeptide marker or markers
is
measured. The treatment may comprise, for example, purification, separation,
dilution or concentration. The methods may be, for example, centrifugation,
filtration, ultrafiltration, dialysis, precipitation or chromatographic
methods, such
as affinity separation or separation by means of ion-exchange chromatography,
or
electrophoretic separation. Particular examples thereof are gel
electrophoresis,
two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), capillary
electro-
phoresis, metal affinity chromatography, immobilized metal affinity
chromatogra-
phy (IMAC), lectin-based affinity chromatography, liquid chromatography, high-
performance liquid chromatography (HPLC), normal and reverse-phase HPLC,
cation-exchange chromatography and selective binding to surfaces. All these
methods are well known to the skilled person, and the skilled person will be
able to
select the method as a function of the sample employed and the method for
determining the presence or absence of the polypeptide marker or markers.
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In one embodiment of the invention, the sample, before being measured, is
separated by capillary electrophoresis, purified by ultracentrifugation and/or
divided by ultrafiltration into fractions which contain polypeptide markers of
a
particular molecular size.
Preferably, a mass-spectrometric method is used to determine the presence or
absence of a polypeptide marker, wherein a purification or separation of the
sample may be performed upstream from such method. As compared to the
currently employed methods, mass-spectrometric analysis has the advantage that
the concentration of many (> 100) polypeptides of a sample can be determined
by
a single analysis. Any type of mass spectrometer may be employed. By means of
mass spectrometry, it is possible to measure 10 fmol of a polypeptide marker,
i.e.,
0.1 ng of a 10 kDa protein, as a matter of routine with a measuring accuracy
of
about 0.01% in a complex mixture. In mass spectrometers, an ion-forming unit
is coupled with a suitable analytic device. For example, electrospray-
ionization
(ESI) interfaces are mostly used to measure ions in liquid samples, whereas
the
matrix-assisted laser desorption/ionization (MALDI) technique is used for
measur-
ing ions from a sample crystallized with a matrix. For analyzing the ions
formed,
quadrupoles, ion traps or time-of-flight (TOF) analyzers may be used.
In electrospray ionization (ESI), the molecules present in solution are
atomized,
inter alia, under the influence of high voltage (e.g., 1-8 kV), which forms
charged
droplets that become smaller from the evaporation of the solvent. Finally, so-
called
Coulomb explosions cause the formation of free ions, which can then be
analyzed
and detected.
In the analysis of the ions by means of TOF, a particular acceleration voltage
is
applied which confers an equal amount of kinetic energy to the ions.
Thereafter,
the time that the respective ions take to travel a particular drifting
distance
through the flying tube is measured very accurately. Since with equal amounts
of
kinetic energy, the velocity of the ions depends on their mass, the latter can
thus
be determined. TOF analyzers have a very high scanning speed and therefore
reach a very high resolution.
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Preferred methods for the determination of the presence and absence of polypep-
tide markers include gas-phase ion spectrometry, such as laser desorption/
ionization mass spectrometry, MALDI-TOF MS, SELDI-TOF MS (surface-enhanced
laser desorption/ionization), LC-MS (liquid chromatography/mass spectrometry),
2D-PAGE/MS and capillary electrophoresis-mass spectrometry (CE-MS). All
methods mentioned are known to the skilled person.
A particularly preferred method is CE-MS, in which capillary electrophoresis
is
coupled with mass spectrometry. This method has been described in some
detail, for example, in the German Patent Application DE 10021737, in Kaiser
et
al. (J Chromatogr A, 2003, Vol. 1013: 157-171, and Electrophoresis, 2004, 25:
2044-2055) and in Wittke et at. (Journal of Chromatography A, 2003, 1013:
173-181). The CE-MS technology allows to determine the presence of some
hundreds of polypeptide markers of a sample simultaneously within a short time
and in a small volume with high sensitivity. After a sample has been measured,
a pattern of the measured polypeptide markers is prepared. This pattern can be
compared with reference patterns of sick or healthy subjects. In most cases,
it is
sufficient to use a limited number of polypeptide markers for the diagnosis of
prostate cancer and the differential diagnosis between prostate cancer and
BPH.
A CE-MS method which includes CE coupled on-line to an ESI-TOF MS device is
further preferred.
For CE-MS, the use of volatile solvents is preferred, and it is best to work
under
essentially salt-free conditions. Examples of suitable solvents include
acetoni-
trile, methanol and the like. The solvents can be diluted with water or
admixed
with a weak acid (e.g., from 0.1% to 1% formic acid) in order to protonate the
analyte, preferably the polypeptides.
By means of capillary electrophoresis, it is possible to separate molecules by
their charge and size. Neutral particles will migrate at the speed of the
electro-
osmotic flow upon application of a current, while cations are accelerated
towards
the cathode, and anions are delayed. The advantage of capillaries in electro-
phoresis resides in their favorable ratio of surface to volume, which enables
a
good dissipation of the Joule heat generated during the current flow. This in
turn
CA 02701571 2010-04-01
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allows high voltages (usually up to 30 kV) to be applied and thus a high
separat-
ing performance and short times of analysis.
In capillary electrophoresis, silica glass capillaries having inner diameters
of
typically from 50 to 75 pm are usually employed. The lengths employed are from
30 to 100 cm. In addition, the capillaries are usually made of plastic-coated
silica glass. The capillaries may be both untreated, i.e., expose their
hydrophilic
groups on the interior surface, or coated on the interior surface. A
hydrophobic
coating may be used to improve the resolution. In addition to the voltage, a
pressure may also be applied, which typically is within a range of from 0 to 1
psi.
The pressure may also be applied only during the performance or altered
meanwhile.
In a preferred method for measuring polypeptide markers, the markers of the
sample are separated by means of capillary electrophoresis, then directly
ionized
and transferred on-line to a mass spectrometer coupled thereto for detection.
In the method according to the invention, it is advantageous to use several
polypeptide markers for the diagnosis of prostate cancer. In particular, at
least
three polypeptide markers may be used, for example, markers 1, 2 and 3; 1, 2
and 4; etc.
More preferred is the use of at least 4, 5 or 6 markers.
Even more preferred is the use of at least 11 markers, for example, markers 1
to
11.
Preferably, a subgroup of the markers are measured, wherein the markers are
selected in such a way as to be found in the sample with a high probability.
Depending on the frequencies of the markers, they are sel4ected in such a way
that at least half the markers are found in a sample with 90% probability. For
example, if only 2 markers are analyzed, for example, markers 1 and 2 in a
patient
with a pathological prostate gland, then the probability that none of the
markers is
found in the sample is 12%, i.e., at least 1 marker is found with 88%
probability.
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Therefore, it is more advantageous to use markers 2 and 3. In this case, the
probability that no marker is found is only about 1%, i.e., the probability
that at
least one of the markers is found is almost 99%. It is known to the skilled
person
how to use statistical methods to select suitable marker combinations, wherein
preferably at least 6, more preferably at least 10, even more preferably at
least 20
markers are analyzed.
Most preferred is the use of all markers listed in Tables 1 or 3.
Several markers may also be used for the differential diagnosis between PCA
and
BPH. In particular, at least three polypeptide markers can be used.
More preferred is the use of at least 4, 5 or 6 markers.
Even more preferred is the use of at least 7 markers.
Most preferred is the use of all the markers listed in Tables 2 or 4.
In order to determine the probability of the existence of prostate cancer when
several markers are used, statistic methods known to the skilled person may be
used. For example, the Random Forests method described by Weissinger et al.
(Kidney Int., 2004, 65: 2426-2434) may be used by using a computer program
such as S-Plus.
In the natural ageing process of humans, the glomerular filtration performance
of the kidney decreases (J. Gerontol. 31 (1976) 155), which results in a
higher
abundance of larger proteins in the urine. This process, which may exhibit a
large variability among individuals in terms of its intensity, complicates the
comparability of measurements for the determination of pathological changes of
the prostate gland from urine. For this reason, the sample preparation of
urine
samples had to be improved to remove these disturbing molecules. The im-
proved protocol removes all molecules larger than 20 kDa, which results in a
greatly improved mutual comparability of the urine samples.
CA 02701571 2010-04-01
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Example:
1. Sample preparation
For the subsequent CE-MS measurement, the proteins which are also contained in
urine in a higher concentration, such as albumin and immunoglobulins, had to
be
separated off by ultrafiltration. Thus, 700 pl of urine was removed and
admixed
with 700 pl of filtration buffer (2 M urea, 10 mM ammonia, 0.02% SDS). This
1.4 ml of sample volume was ultrafiltrated (20 kDa, Sartorius, Gottingen, DE).
The ultrafiltration was performed at 3000 rpm in a centrifuge until 1.1 ml of
ultrafiltrate was obtained. The 1.1 ml of filtrate obtained was then applied
to a PD
column (Amersham Bioscience, Uppsala, Sweden), and desalted against
2.5 ml of 0.01% NH4OH and lyophilized. For the CE-MS measurement, the
polypeptides were then resuspended with 20 pl of water (HPLC grade, Merck).
2. CE-MS measurement
The CE-MS measurements were performed with a capillary electrophoresis system
from Beckman Coulter (P/ACE MDQ System; Beckman Coulter Inc., Fullerton,
USA) and an ESI-TOF mass spectrometer from Bruker (micro-TOF MS, Bruker
Daltonik, Bremen, Germany).
The CE capillaries were supplied by Beckman Coulter and had an ID/OD of
50/360 pm and a length of 90 cm. The mobile phase for the CE separation
consisted of 20% acetonitrile and 0.25% formic acid in water. For the "sheath
flow" on the MS, 30% isopropanol with 0.5% formic acid was used at a flow rate
of 2 pl/min. The coupling of CE and MS was realized by a CE-ESI-MS Sprayer Kit
(Agilent Technologies, Waldbronn, Germany).
For injecting the sample, a pressure of from 1 to a maximum of 6 psi was
applied, and the duration of the injection was 99 seconds. With these parame-
ters, about 150 nl of the sample was injected into the capillary, which corres-
ponds to about 10% of the capillary volume. A stacking technique was used to
concentrate the sample in the capillary. Thus, before the sample was injected,
a
CA 02701571 2010-04-01
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1 M NH3 solution was injected for 7 seconds (at 1 psi), and after the sample
was
injected, a 2 M formic acid solution was injected for 5 seconds. After the
separation voltage (30 kV) was applied, the analytes were automatically
concentrated between these solutions.
The subsequent CE separation was performed with a pressure method: 40
minutes at 0 psi, then 0.1 psi for 2 min, 0.2 psi for 2 min, 0.3 psi for 2
min,
0.4 psi for 2 min, and finally 0.5 psi for 32 min. The total duration of a
separa-
tion run was thus 80 minutes.
In order to obtain as good as possible a signal intensity on the side of the
MS,
the nebulizer gas was set to the lowest possible value. The voltage applied to
the
spray needle for generating the electrospray was 3700-4100 V. The remaining
settings at the mass spectrometer were optimized for peptide detection accord-
ing to the manufacturer's protocol. The spectra were recorded over a mass
range of m/z 400 to m/z 3000 and accumulated every 3 seconds.
In addition to the modified sample preparation, the comparability of the
individ-
ual urine samples was improved by calibrating the masses and migration times.
For calibration, a "local linear regression" with reference polypeptides as
known
to the skilled person was performed. Any polypeptides defined by an exact mass
and migration time can be used as reference proteins, and polypeptides having
a
high signal-to-noise ratio are preferably used, "house keeping proteins" being
more preferably used. "House keeping proteins" are protein standards that are
naturally present in the majority of the urine samples in slightly varying
amounts.
3. Standards for the CE measurement
For checking the CE measurement and calibrating, the following proteins or
polypeptides which are characterized by the stated CE migration times under
the
selected conditions were employed:
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Protein/polypeptide Migration time
Aprotinin (SIGMA, Taufkirchen, DE, Cat. # A1153) 19.3 min
Ribonuclease (SIGMA, Taufkirchen, DE, Cat. # R4875) 19.55 min
Lysozyme (SIGMA, Taufkirchen, DE, Cat. # L7651) 19.28 min
"REV", Sequence: REVQSKIGYGRQIIS 20.95 min
"ELM", Sequence: ELMTGELPYSHINNRDQIIFMVGR 23.49 min
"KINCON", Sequence: TGSLPYSHIGSRDQIIFMVGR 22.62 min
"GIVLY" Sequence: GIVLYELMTGELPYSHIN 32.2 min
The proteins/polypeptides were employed at a concentration of 10 pmol/pl each
in water. "REV", "ELM, "KINCON" and "GIVLY" are synthetic peptides.
The above described modifications resulted in an improvement over WO
03/072710 A2 in view of both the detection limits and the reproducible detecta-
bility of polypeptides. Thus, polypeptides could be identified that indicate
pathological alterations of the prostate gland (Pca (prostate cancer), high
grade
PIN (prostatic intra-epithelial neoplasia), BPH (benign prostate hyperplasia)
and
ASAP (atypical small acinar proliferation)) as compared to a healthy,
physiologi-
cal prostate gland (Table 1). In addition, due to these improvements, polypep-
tides could be identified that allow a distinction between PCa and BPH that is
improved as compared to WO 03/072710 A2 (Table 2).
The molecular masses of the peptides and the m/z ratios of the individual
charge
states visible in MS are listed in the following Table:
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H 1.0079 1.0079 1.0079 1.0079 1.0079 1.0079 1.0079
(mono)
m/z Aprotinin Ribonuclease Lysozyme REV KINCON ELM GIVLY
Mono Mono Mass Mono Mass Mono Mono Mono Mono
Mass Mass Mass Mass Mass
0 6513.09 13681.32 14303.88 1732.96 2333.19 2832.41 2048.03
1 6514.0979 13682.328 14304.888 1733.9679 2334.1979 2833.4179 2049.0379
2 3257.5529 6841.6679 7152.9479 867.4879 1167.6029 1417.2129 1025.0229
3 2172.0379 4561.4479 4768.9679 578.6612 778.7379 945.1446 683.6846
4 1629.2804 3421.3379 3576.9779 434.2479 584.3054 709.1104 513.0154
1303.6259 2737.2719 2861.7839 347.5999 467.6459 567.4899 410.6139
6 1086.5229 2281.2279 2384.9879 289.8346 389.8729 473.0762 342.3462
7 931.4494 1955.4822 2044.4193 248.5736 334.3208 405.6379 293.5836
8 815.1442 1711.1729 1788.9929 217.6279 292.6567 355.0592 257.0117
9 724.6846 1521.1546 1590.3279 193.559 260.2512 315.7201 228.5668
652.3169 1369.1399 1431.3959 174.3039 234.3269 284.2489 205.8109
11 593.107 1244.7643 1301.3606 158.5497 213.1161 258.4997 187.1924
12 543.7654 1141.1179 1192.9979 145.4212 195.4404 237.0421 171.6771
13 502.0148 1053.4171 1101.3063 134.3125 180.4841 218.8856 158.5486
Performance of the markers described in WO 01/25791
WO 01/25791 mentions eleven markers (see claim 2 thereof), and the accuracy of
mass determination is stated to be 0.5%. If it is considered that these are
only
fragments of semenogelin I, the following numbers of possible fragments of
semenogelin are obtained:
CA 02701571 2010-04-01
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Molecular masses of the biomarker Number of possible fragments from
seminogelin I
polypeptides for prostate carcinoma Mass deviation 0.5 % Mass deviation 0.03%
[glmol]
2776 100 8
4423 182 16
4480 173 8
5753 212 11
6098 214 7
6270 217 19
6998 248 12
7843 270 11
8030 265 23
8240 281 12
8714 299 23
Further, WO 01/25791 discloses markers that speak against the existence of a
prostate carcinoma. The number of possible fragments is contained in the
following
Table:
Number of possible fragments from seminogelin I
Molecular masses of the
biomarker polypeptides for Mass deviation 0.5 % Mass deviation 0.03%
prostate carcinoma [g/mol]
2095 83 2
2276 84 5
2530 99 6
3030 106 4
3038 116 10
3224 127 7
3600 139 6
3835 146 9
3915 150 16
3933 153 23
4175 161 14
Illustratively for one mass, Figure 1 shows the large number of sequences that
are
possible in this weight range.
In the next step, it was tried to analyze the corresponding proteins in urine
samples from patients with prostate carcinoma or BPH. Only six markers could
be
found.
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Figure 2 shows a determination of the amplitudes of the six markers found in
this
way. It is found that no significant differences occur between prostate
carcinoma
patients and the control group. Subsequently, the discriminatory value of the
biomarkers was examined by means of an ROC (receiver operator characteristic
curves) analysis.
Figure 3a shows corresponding analyses for six markers that are described in
WO
01/25791 and could be found in urine samples.
Figure 3b shows a corresponding ROC examination of the biomarkers according to
the invention with Nos. 142 to 201 of the present patent application. Figure
3c
shows the ROC analysis of a subgroup of the markers according to the invention
(12 markers).
Figure 3d shows that the significance is clearly higher than that of WO
01/25791
even if only three biomarkers according to the invention were used.
